Interaction of L-canaline with ornithine aminotransferase of the tobacco hornworm, Manduca sexta (Sphingidae).
Ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase (EC 2.6.1.13)) has been purified to homogeneity from last instar larvae of the tobacco hornworm, Manduca sexta (Sphingidae). This enzyme is a 144,000-Da tetramer constructed from 36,000-Da protomeric units. It has a high aspartate/asparagine and glutamate/glutamine content and 2 cysteine residues/subunit. All 8 cysteine residues can react with N-ethylmaleimide to inactivate the enzyme. Maintenance of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol maximizes enzymatic activity and improves storage conditions, presumably by protecting these sulfhydryl groups. The apparent Km values for L-ornithine and 2-oxoglutaric acid are 2.3 and 3.2 mM, respectively. The turnover number is 2.0 +/- 0.1 mumol min-1 mumol-1. L-Canaline (L-2-amino-4-(aminooxy)butyric acid) is a potent ornithine aminotransferase inhibitor. Reaction of the enzyme with L-[U-14C]canaline produces an enzyme-bound, covalently linked, radiolabeled canaline-pyridoxal phosphate oxime. The L-[U-14C]canaline-pyridoxal phosphate oxime has been isolated from canaline-treated enzyme. Dialysis of canaline-inactivated ornithine aminotransferase against free pyridoxal phosphate slowly reactivates the enzyme as the oxime is replaced by pyridoxal phosphate. Analysis of L-[U-14C]canaline binding to ornithine aminotransferase reveals the presence of 4 mol of pyridoxal phosphate/mol of enzyme.
Rosenthal
G A
GA
Graduate Center for Toxicology, University of Kentucky, Lexington 40506.
Dahlman
D L
DL
D000596:Amino Acids / Q000032:analysis; D000613:Aminobutyrates / Q000378:metabolism*; D000723:Ancylostomatoidea / Q000201:enzymology*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D009953:Ornithine-Oxo-Acid Transaminase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000032:analysis; D013050:Spectrometry, Fluorescence; D000637:Transaminases / Q000378:metabolism*
D000596:Amino Acids; D000613:Aminobutyrates; C001926:canaline; D011732:Pyridoxal Phosphate; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase; D005033:Ethylmaleimide